PROJECT SUMMARY Central to the pathogenesis of many bacteria are sophisticated secretion systems that translocate proteins from the cytosol of bacteria into target eukaryotic cells. Despite a great deal of interest, there remains a fundamental gap in our understanding of the molecular function of most bacterial effectors. This gap represents an important problem because the virulence of intracellular bacterial pathogens relies on these proteins; therefore, understanding how individual effectors contribute to bacterial replication and survival is key to understanding mechanisms of pathogenesis and developing effective therapeutics. The long-term goal of this project is to elucidate how Salmonella enterica serovar Typhimurium (STm) effectors modulate endocytic trafficking through manipulation of the host retromer complex. The retromer is a key component of the endosomal protein sorting machinery and mostly functions to recognize and sort cargo from the canonical endocytic pathway to either the plasma membrane or the Golgi network. Subversion of retrograde transport is emerging as an important adaptation for intracellular pathogen survival, with the retromer being a favorite target of translocated bacterial effectors. The central hypothesis of this proposal is that direct engagement of the retromer by the STm effector SseC is required for maintaining the Salmonella-containing vacuole and establishing of a replicative niche in host cells. An innovative high-throughput yeast genetic screen identified the retromer as a potential target of SseC. Although strong follow-up experiments clearly show that SseC directly interacts with components of the human retromer complex (VPS35 and VPS26A) with nanomolar affinity, why this interaction is important for STm survival remains unclear. As ?sseC Salmonella are completely avirulent in a mouse model of infection, modulation of the retromer may be a crucial component of Salmonella pathogenesis. This proposal pursues two specific aims: 1) Demonstrate a role for the retromer in determining the outcome of STm infection; and 2) Determine the structure of the SseC:retromer complex and translate knowledge of the SseC-retromer interface to structure-function analysis during STm infection. The first aim combines live-cell imaging and the generation of retromer knockout human cell lines with STm infection to determine how modulation of the retromer affects STm trafficking and survival in infection-relevant cell types. The second aim leverages the already solved crystal structure of the retromer complex to probe the SseC:retromer interface at the atomic level and uses this structure to inform structure-function experiments in cells. The approach is innovative in its application of our novel yeast screen to characterize SseC mutants and in that no one has implicated the retromer complex in STm infection to date. The proposed research is significant because it will illuminate mechanisms that bacteria have evolved to subvert or co-opt the retromer, thus contributing to the design of host-directed therapeutics that may be effective against phylogenetically diverse bacterial pathogens. !